Multiplex Fluorescent In Situ Hybridization in Zebrafish Embryos Using Tyramide Signal Amplification
Multiplex Fluorescent In Situ Hybridization in Zebrafish Embryos Using Tyramide Signal Amplification
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DOI：
10.1089/zeb.2005.2.105
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发表时间：
2005-01-01
期刊：
影响因子：
2
通讯作者：
Ramakrishnan, Lalita
中科院分区：
文献类型：
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作者：
Clay, Hilary;Ramakrishnan, Lalita
斑马鱼的优势之一是可以轻松地进行原位杂交以确定整个胚胎中基因表达的空间和时间模式。迄今为止，比色检测方法主要用于这些分析。在这里，我们描述了使用酪酰胺信号放大（TSA）的全镶嵌斑马鱼胚胎的荧光原位杂交（FISH）方案。确定了一组最佳试剂，可以同时定位同一胚胎内两个基因的基因表达模式，从而可以识别单细胞内的共定位表达。该协议可以扩展为通过使用不同的荧光染料标签按顺序重复每个目标的基于 TSA 的检测来执行多重研究。为此，我们证明除了 FISH 之外，该方法还可以与标准辣根过氧化物酶 (HRP) 介导的免疫细胞化学程序相结合。
One of the strengths of the zebrafish is the ease with which in situ hybridization can be performed to determine spatial and temporal patterns of gene expression in whole embryos. Thus far, colorimetric detection methods are mainly used for these analyses. Here we describe a fluorescent in situ hybridization (FISH) protocol for whole-mount zebrafish embryos using tyramide signal amplification (TSA). An optimal set of reagents was identified that allows for simultaneous localization of gene expression patterns of two genes within the same embryo, permitting identification of colocalized expression within single cells. This protocol can be extended to perform multiplex studies by repetition of the TSA-based detection for each target sequentially with a different fluorescent dye label. To this effect, we demonstrate that this approach can be combined with standard horseradish peroxidase (HRP)-mediated immunocytochemistry procedures in addition to FISH.